Human proteins made in yeast

Researchers in the US have managed for the first time to produce a fully human protein - including complex sugar side chains - in a yeast organism. The work is reported in the journal Science​.

Yeasts and other fungi are reliable, cost-effective workhorses for the production of many industrial enzymes, but biopharmaceutical companies currently manufacture few human therapeutic proteins using these organisms. Now, researchers in the USA have overcome the key obstacle to the widespread adoption of yeast as a production vehicle for human proteins, i.e. the inability to produce human proteins with complex sugar molecules - a process known as glycosylation.

The scientists, from GlycoFi​ and Dartmouth College, have successfully re-engineered the glycosylation pathway in the yeast Pichia pastoris​ to allow the creation of a fully-human protein, extending work published earlier this year which resulted in a hybrid​ protein.

Dr Stephen Hamilton, a GlycoFi scientist and the lead author of the publication, said: "Mammalian cell lines can replicate human-like glycoprotein processing to a large extent, and so have traditionally been used to produce most protein therapeutics. However, mammalian cell culture systems have significant drawbacks including low protein yields, long fermentation times, production of a mixture of protein glycoforms, and ongoing viral contamination issues."​

The ability to produce human glycoproteins with homogenous N-glycan structures provides access to the inherent commercial advantages of yeast and other fungal production systems, said Tillman Gerngross, GlycoFi's chief scientific officer and associate professor of biochemical engineering at Dartmouth College. "Moreover, the ability to produce a homogeneous glycoprotein in yeast offers the biopharmaceutical industry a new tool for further understanding the structure-function relationships of glycoproteins and for potentially creating safer, more effective therapeutics."​

P. pastoris​ is already commonly used in fermentation processes and can be grown to high cell density in a chemically defined growth medium. This yeast normally produces non-human N-glycans of the high mannose type, which have no therapeutic value for humans. The scientists modified the yeast by first eliminating endogenous yeast glycosylation pathways, while sequentially engineering into the organism five active eukaryotic proteins, including mannosidases I & II, N-acetylglucosaminyl transferases I & II and UDP-N-acetylglucosamine transporter.

The targeted localisation of these enzymes enabled the generation of a synthetic in vivo​ glycosylation pathway that enabled the yeast to produce a complex human N-glycan, GlcNAc2Man3GlcNAc2. However, unlike the glycosylation pathway in mammalian cell lines, which typically produces an array of glycoforms, the genetically modified yeast yielded essentially homogeneous glycoforms.

"We have essentially been able to humanise the yeast, where it is able to make a single glycoform of exceptional uniformity,"​ Dr Gerngross said. Using mammalian cell culture it is difficult to isolate individual protein glycoforms and even more difficult to produce specific structures at a commercial scale.

"The ability to express a single protein in a library of genetically engineered yeasts - each producing a defined and uniform glycoform - will enable the generation of glycoprotein libraries that can be used both to elucidate specific structure-function relationships and to identify the most efficacious molecule for a particular therapeutic use. Moreover, once identified, a particular protein glycoform can be readily produced at industrial scale using the relevant yeast, due to the well-established rapidity with which yeast fermentations can be scaled up,"​ said Dr Gerngross.